Background/Purpose: Rheumatoid arthritis (RA) is an incurable autoimmune disease where response to therapy is heterogenous. Clinically indistinguishable RA patients can be classified histo-pathologically into three distinct pathotypes: lympho-myeloid (LM), diffuse-myeloid (DM) and fibroid-pauci-immune (FPI). They are characterized by heavy lymphocytic infiltration (LM), macrophage rich / lymphoid poor (DM) and fibroblast rich / myeloid and lymphoid poor (FPI). Understanding the signaling pathways associated with these pathotypes is likely to be critical for therapeutic targeting. Our pilot (phospho)proteome analysis of 8 early-stage RA synovial biopsies highlighted that immunological pathways were preferentially associated with LM compared to DM with distinct phosphorylated kinases¹. Here, to establish the dynamics of expression across disease stages, we expanded that analysis to advanced, anti-TNF inadequate responder RA patients recruited to the R4RA trial^2,3.  

Methods: The phosphoproteome and proteome profiles of 33 pre-treatment synovial biopsies from the R4RA trial were analyzed using label-free mass spectrometry (Figure 1A). There were 21 lympho-myeloid, 7 diffuse-myeloid and 5 fibroid-pauci-immune samples.  

Results: Principal component analysis highlighted that synovial pathotypes are key drivers of the phosphoproteome and proteome profiles (Figure 1B-C). Lympho-myeloid was the most immunologically active with the upregulation of lymphoproliferative signaling pathways, recapitulating the pilot study with lymphoid and myeloid samples¹. This larger scale analysis demonstrated an enrichment of B cell receptor related kinases, such as B Lymphocyte Kinase (BLK, p = 0.001) in LM compared to DM pathotype. Furthermore, the gradient of immune signaling across the three pathotypes (LM > DM > FPI) is also seen through the expression of Mammalian Target of Rapamycin (MTOR) where MTOR and 3-Phosphoinositide Dependent Protein Kinase 1 (PDPK1) were upregulated in LM compared to FPI (p = 0.001 and 0.009, respectively), whereas PDPK1 was enriched in DM compared to FPI (p = 0.008). Conversely, the FPI (phospho)proteome signatures were associated with cytoskeletal and extracellular matrix processes (e.g., vimentin and collagen proteins), consistent with known fibroblast function.

Conclusion: Synovium (phospho)proteome analysis identified specific signaling pathways associated with distinct pathotypes both in early and advanced RA, which can be exploited for targeted therapeutic applications. R4RA treatment response analysis is ongoing.

References 
¹Çubuk, C. et al. (2024), Arthritis Research & Therapy, 26(1). doi:10.1186/s13075-024-03351-4. 
²Humby, F. et al. (2021), The Lancet, 397(10271), pp. 305–317. doi:10.1016/s0140-6736(20)32341-2. 
³Rivellese, F. et al. (2022), Nature Medicine, 28(6), pp. 1256–1268. doi:10.1038/s41591-022-01789-0. 

Acknowledgements
https://www.nature.com/articles/s41591-022-01789-0#Ack1

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/synovial-phosphoproteomic-profiling-in-early-and-advanced-rheumatoid-arthritis-r4ra-trial-identifies-specific-signaling-pathways-linked-to-distinct-pathotypes/